Engineered cxcl12 alpha locked dimer polypeptide

ABSTRACT

The present invention provides a novel CXCL12-α 2  locked dimer polypeptide, pharmaceutical compositions thereof, and methods of using said dimer in the treatment of cancer, inflammatory disorders, autoimmune disease, and HIV/AIDS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/936,650, filed on Jul. 8, 2013, which is a divisional of U.S.application Ser. No. 12/956,514, filed on Nov. 30, 2010 and issued asU.S. Pat. No. 8,524,670 on Sep. 3, 2013, which is a continuation of U.S.application Ser. No. 12/380,308, filed on Feb. 26, 2009 and issued asU.S. Pat. No. 7,923,016 on Apr. 4, 2011, which claims priority to U.S.Provisional Application No. 61/067,273 filed Feb. 27, 2008. Each ofthese applications and patents is incorporated by reference herein inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI058072 awardedby the National Institutes of Health-NIAID. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates generally to a novel CXCL12-α₂ locked dimerpolypeptide, pharmaceutical compositions thereof, and methods of usingsaid dimer in the treatment of cancer and autoimmune, inflammatorydisease and HIV/AIDS.

BACKGROUND

Chemokines are a superfamily of chemoattractant cytokine proteins whichprimarily serve to regulate a variety of biological responses andpromote the recruitment and migration of multiple lineages of leukocytesand lymphocytes to a body organ tissue. Chemokines are classified intofour families according to the relative position of the first twocysteine residues in the protein. In one family, the first two cysteinesare separated by one amino acid residue (the CXC chemokines) and inanother family the first two cysteines are adjacent (the CC chemokines).In a third family, the first two cysteines are separated by three aminoacids (CX₃C chemokines). In a fourth family there is only one cysteinein the amino terminus (C chemokines).

The molecular targets for chemokines are cell surface receptors. Onesuch receptor is CXC chemokine receptor 4 (CXCR4), which is a seventransmembrane G-protein coupled receptor (GPCR). CXCR4 is widelyexpressed on cells of hematopoietic origin, and is a major co-receptorwith CD4⁺ for certain strains of human immunodeficiency virus 1 (HIV-1).

CXCL12, formerly known as stromal cell-derived factor-1 (SDF-1), is analpha or CXC type 7.8 kDa CXC chemokine. CXCL12 is the only knownnatural ligand for CXCR4, as high affinity CXCL12 binding requires theCXCR4 amino terminus. CXCL12 comprises two closely related members:CXCL12-α and CXCL12-β, the native amino acid sequences of which areknown, as are the genomic sequences encoding these proteins (U.S. Pat.No. 5,563,048 and U.S. Pat. No. 5,756,084, both of which areincorporated by reference herein for all purposes).

Originally described as a growth factor for bone marrow developing Bcells, CXCL12 was subsequently characterized as a chemoattractant for Tcells and monocytes. Genetic ablation of CXCR4 or CXCL12 results indefects in haematopoiesis, vascularization of the intestines, cerebellarformation and heart development. Similar embryonic defects in either ofthose chemokine receptor or chemokine gene deficient animals hasrevealed roles for CXCR4-CXCL12 signaling in cardiovascular, neuronal,and hematopoietic stem cell development as well as gastrointestinalvascularization. Previous studies have also established a role forCXCL12 and CXCR4 in gut vascularization, a key process in mucosalimmunity and homeostasis. In vitro, CXCL12 stimulates chemotaxis of awide range of cells including monocytes and bone marrow derivedprogenitor cells. Particularly notable is its ability to stimulate ahigh percentage of resting and activated T-lymphocytes.

Consistent with the fact that CXCR4 is a major co-receptor for HIV,CXCL12 has also been shown to block HIV entry into CD4+ cells. CXCR4 isa co-receptor for T-tropic (X4) strains of HIV, which target CD4⁺ Tcells, and CXCL12 can inhibit HIV-1 infection by preventing gp120binding to CXCR4 and the subsequent gp41 mediated fusion. CXCR4co-receptor usage correlates with AIDS onset, even though CCR5 is theprimary co-receptor for most HIV infections.

Efforts have been made to identify CXCL12-derived peptides thatinterfere selectively with HIV entry, and not with CXCL12 signaling. Awide range of potential CXCR4 binding fragments of CXCL12 have beenproposed for use in blocking HIV infection, indicating that the anti-HIVactivity of CXCL12, or fragments of CXCL12, does not depend onantagonism of the CXCR4 receptor.

CXCL12 also directs homeostatic immune cell trafficking and inflammatoryresponses. Chemokine activation of specific G-protein coupled receptors(GPCR) directs cell migration toward higher chemokine concentration.

Additionally, CXCL12 and CXCR4 mediate cancer cell migration andmetastasis. Treatment with CXCR4-neutralizing antibodies reducedmetastatic tumor formation in a mouse model for human breast cancer.Subsequently, over twenty cancer types have been shown to express CXCR4and metastasize to tissues that secrete CXCL12, such as bone marrow,lung, liver and lymph nodes.

CXCL12 and CXCR4 also serve to establish a niche environment forhematopoetic stem cells in bone marrow such that blocking the functionof CXCL12 leads to mobilization of said stem cells so that they exit thebone marrow and enter the blood stream.

Accordingly, there is a current need for cost-effective pharmaceuticalagents and treatment methods for treating various conditions includingautoimmune or inflammation disorders, immune suppression conditions,infections, blood cell deficiencies, cancers and other describedconditions and to mobilize stem cells by manipulating and controllingCXCL12 and CXCR4.

SUMMARY OF THE INVENTION

The inventors have engineered a novel CXCL12-α₂ locked dimer polypeptidecomprising two monomers linked together. The dimer will/might be usefulin treating various conditions including cancer, autoimmune disordersand/or inflammation disorders. In one preferred embodiment the dimercomprises two monomers bound together, wherein at least one monomer hasthe amino acid sequence as shown in SEQ ID NO:1.

In another embodiment, the present invention provides a compositioncomprising a CXCL12-α₂ locked dimer polypeptide and a pharmaceuticallyacceptable carrier or diluent.

In another embodiment, the present invention provides an isolatedCXCL12-α₂ locked dimer polypeptide, wherein the dimer preferablyconsists of at least one monomer having the amino acid sequence as shownin SEQ ID NO:1.

In another embodiment, the present invention provides a method oftreating an autoimmune disease in a subject comprising administering tothe subject a therapeutically effective amount of a compositioncomprising a CXCL12-α₂ locked dimer.

In another embodiment, the present invention provides a method oftreating a tumor in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide.

In another embodiment, the present invention provides a method oftreating HIV/AIDS in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide.

In another embodiment, the present invention provides a method ofinhibiting angiogenesis in a subject by administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide.

In another embodiment, the present invention provides a method ofinhibiting blood cancers in a subject by administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide.

In another embodiment, the present invention provides a kit comprising aCXCL12-α₂ locked dimer polypeptide wherein the dimer preferablycomprises at least one monomer having the amino acid sequence as shownin SEQ ID NO:1, a pharmaceutically acceptable carrier or diluent, andinstructional material.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. CXCL12-α₂ (L36C/A65C) (CXCL12-α₂) is a covalently locked dimercomprising two CXCL12 L36C/A65C sequences (SEQ ID NO: 1), each sequencecomprising one subunit of the dimer. The lines connecting the cysteinesshow where the intra- and intermolecular disulfide bonds are. A)CXCL12-α₂ locked dimer amino acid sequence with the conservedintramolecular disulfide bonds (black lines) and the engineeredintermolecular disulfide bonds (red lines) illustrated. B) SDS-PAGE ofCXCL12-α and CXCL12-α₂ treated with and without dithiothreitol (DTT).CXCL12-α and CXCL12-α₂ migrate near the monomeric molecular weight of 8kDa when treated with DTT. In contrast, CXCL12-α₂ migrates as a dimerwhile CXCL12-α migrates as a monomer in the absence of DTT. C)Translational diffusion measurements of CXCL12-α₂ indicate CXCL12-α₂ isdimeric. Diffusion coefficients (D_(s)) of wild-type CXCL12 (blackcircles) in 20 mM sodium phosphate at pH 7.4 plotted versus chemokineconcentration. Non-linear fitting of the CXCL12-α D_(s) values indicatesa dimer dissociation K_(d) of 120 μM with a pure monomer D_(s) value of1.6 (×10⁻⁶ cm²s⁻¹) and a dimer value of ˜1.0 (×10⁻⁶ cm²s⁻¹). D_(s)values for 10, 50, and 150 μM SDF1₂ (red triangles) range from 1.08-1.09(×10⁻⁶ cm²s⁻¹) consistent with those expected for CXCL12-α in thedimeric state. D) N-terminal peptides corresponding to the firstthirty-eight amino acids of CXCR4 are illustrated. The sequence for p38is identical to that of CXCR4 except for an additional N-terminalgly-ser dipeptide (cloning artifact) and the C28A substitution (green)introduced to prevent oxidative peptide dimer formation. The sulfatedpeptides are identical to p38 except for the inclusion of sulfotyrosineat position 21 for p38-sY₁ and at positions 7, 12 and 21 for p38-sY₃.

FIG. 2. CXCR4 N-terminus binds CXCL12-α₂ (L36C/A65C) (CXCL12-α₂). A)Ensemble of 20 CXCL12-α₂ NMR solution structures (gray and tan)superimposed on the crystal structure of dimeric wild-type CXCL12-α(blue, PDB ID 2J7Z) with an α-carbon RMSD of 1.2 Å for residues 9-66.Intermolecular C36-C65 disulfide bonds are shown in yellow. FlexibleN-terminal residues of CXCL12-α (residues 1-8) are omitted for clarity.Refinement statistics for the CXCL12-α₂ structure ensemble are given intable S1. B) ¹⁵N-¹H HSQC spectra of 25 μM [U-¹⁵N]-CXCL12-α₂ alone (blackcontours) and after addition of 100 μM p38 peptide (green contours). C)Combined ¹⁵N-¹H chemical shift perturbations plotted versus CXCL12-α₂residue number. Secondary structure elements are indicated and regionsinvolved in the dimer interface are highlighted in orange. Missingvalues correspond to prolines (sequence positions 2, 10, 32 and 53) oramino acids not observed in the ¹⁵N-¹H HSQC spectra.

FIG. 3. CXCL12-α₂ CXCR4 N-terminal domain structures. (A) Representativeintermolecular NOEs for the CXCL12-α₂:p38-sY₁ complex. Strips from 3DF1-¹³C-filtered/F3-¹³C-edited NOESY-HSQC spectra acquired on a complexcontaining [U-¹⁵N, ¹³C]-CXCL12-α₂ and unlabeled p38-sY₁ (left) and acomplex containing [U-¹⁵N, ¹³C]-p38-sY₁ and unlabeled CXCL12-α₂ (right)contain equivalent NOEs between the V18 methyl of SDF1₂ and sY21 ¹H^(δ)of p38-sY₁. Ensembles of the twenty lowest energy conformers for the (B)CXCL12-α₂:p38, (C) CXCL12-α₂:p38-sY₁, and (D) CXCL12-α₂:p38-sY₃complexes. CXCL12-α₂ is shown in gray and the CXCR4 N-termini areorange. Sulfotyrosine residues in CXCR4 N-terminus are shown in red.

FIG. 4. Recognition of sulfotyrosine by CXCL12-α₂. A) NMR structure ofCXCL12-α₂ bound to p38-sY₃. Individual monomers of the symmetricCXCL12-α₂ dimer are shown in tan and white with symmetry-related p38-sY₃peptides in blue and orange. Chemical shift perturbations greater than0.25 ppm (FIG. 1C) are highlighted in green on the surface of theCXCL12-α₂ surface. Flexible regions of CXCL12-α₂ (residues 1-8) andp38-sY₃ (residues 29-38) are omitted for clarity. Sulfotyrosine sidechains are shown in ball-and-stick representation. In panels B-D, basicresidues in CXCL12-α₂ that pair with CXCR4 sulfotyrosines are shown inblue and CXCL12-α₂ residues with NOEs to the sulfotyrosines are shown ingreen. B) CXCR4 sY7 binds CXCL12-α₂ near R20 and makes NOE contacts withV23. C) CXCR4 sY12 occupies a cleft bounded by CXCL12-α₂ residues K27,P10 and L29. D) CXCR4 sY21 pairs with CXCL12-α₂ R47 and makes NOEcontacts with V18 and V49.

FIG. 5. Dimeric CXCL12-α₂ induces CXCR4-mediated Ca²⁺-flux but inhibitschemotaxis toward monomeric wild-type CXCL12-α. A) Ca²⁺-flux in THP-1cells loaded with Fluo-3 (Invitrogen) indicates robust dose-dependantCXCR4 activation by wild-type CXCL12-α (, EC₅₀=3.6 nM) and CXCL12-α₂ (

, EC₅₀=12.9 nM). B) Wild-type CXCL12-α chemoattracts THP-1 cells in abiphasic, concentration-dependent manner with maximal migratory responseat ˜30 nM. In contrast, CXCL12-α₂ does not chemoattract THP-1 cells atall protein concentrations from 1-1,000 nM. C) Schematic illustration ofthe bell-shaped profile for CXCL12-α-mediated chemotaxis arising fromchanges in relative concentrations of chemokine monomer and dimer. Atlow chemokine concentration monomeric CXCL12-α promotes chemotaxis(green curve), while increasing CXCL12-α₂ dimerization at high chemokineconcentrations halts chemotaxis (red curve). D) Wild-type CXCL12-α andthe dimerization-impaired H25R variant chemoattract THP-1 cells equallywell at low concentrations (0.1-10 nM). CXCL12-α (H25R) remainsmonomeric at higher concentrations relative to wild-type CXCL12-α andinduces chemotaxis over a broader range. E) Chemoattraction of THP-1cells induced by 10 nM wild-type CXCL12-α is inhibited by CXCL12-α₂(IC₅₀˜4 nM).

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention, which will be limited only byany later-filed nonprovisional applications.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. All publications and patents specificallymentioned herein are incorporated by reference for all purposesincluding describing and disclosing the materials, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

II. The Invention

CXCL12-α₂ Locked Dimer Polypeptide. In one embodiment, the inventionprovides a CXCL12-α₂ locked dimer polypeptide comprising at least twomonomers. The monomers may be identical or may be non-identical. In oneembodiment, at least one of the monomers has the amino acid sequenceaccording to SEQ ID NO: 1. In alternate embodiments, both monomers havethe amino acid sequence according to SEQ ID NO:1.

By “locked” we mean the monomer components of the polypeptide are linkedto each other via at least one covalent bond. The monomer and dimerforms do not interconvert. In a preferred embodiment, at least one ofresidues L36 and A65 are replaced with cysteine residues to create atleast one intermolecular disulfide bond between cysteine residues atposition 36 of one subunit and/or position 65 of the other monomersubunit. As shown in FIG. 1A, either or both cysteine residues atpositions L36 and A65 can be replaced with cysteines to form the lockeddimer with at least one, but preferably two, disulfide bonds.

Other residue(s) besides L36C and A65C in CXCL12 could be mutated tocysteines in order to form the locked dimer similar to the one of thepresent invention. For instance, a locked dimer can be created bymutating amino acid(s) in the CXCL12 dimer interface to cysteines thatare positioned opposite one another yielding a disulfide bond thatcovalently links two CXCL12 monomers. For example, residue K27 isdirectly across the CXCL12 dimer interface from residue K27 of theopposing subunit and K27C mutation would likely make a locked dimer.Residues L26 and I28 are also on the CXCL12 dimer interface, and aL26C/I28C variant should form a locked dimer with L26C of one monomersubunit forming a disulfide bond with I28C of the opposing subunit andI28C of one monomer subunit forming a disulfide bond with L26C of theopposing subunit.

In a preferred embodiment, the CXCL12α₂ locked dimer of the presentinvention has substitutions at both L36C/A65C residues. Residue L36 ison beta strand 2 and A65 is near the end of the alpha helix of thedimer. Thus, disulfide bonds form between beta strand 2 and the end ofthe helix generate the locked dimer. A similar locked dimer could becreated using disulfide bonds introduced between beta strand 1 and themiddle of the alpha helix. For example, CXCL12 with I28C/Y61C orI28C/L62C would form a locked dimer with beta strand one of one monomerhaving a disulfide bond to the middle of the alpha helix of the secondmonomer thus making a locked dimer. Additionally, a locked dimer may becreated by generating a construct that produces two CXCL12 monomerswhere the C-terminus of one is linked to the N-terminus of the otherthrough an amino acid linker.

Additional methods for making locked dimers of CXCL12 could also includeother types of covalent linkages besides disulfide bonds including, butnot limited to, chemical cross-linking reagents.

In a preferred embodiment, the locked dimer of the present inventioncomprises a substantially pure preparation. By “substantially pure” wemean a preparation in which more than 90%, e.g., 95%, 98% or 99% of thepreparation is that of the locked dimer.

In a preferred embodiment, at least one of the monomers comprising thelocked dimer of the present invention has the amino acid sequence asshown in SEQ ID NO:1 or a homologue or fragment thereof. In a furtherpreferred embodiment, the dimer comprises two monomers having the aminoacid sequence as shown in SEQ ID NO:1 or a homologue or variant thereof.By “homologue” we mean an amino acid sequence generally being at least80%, preferably at least 90% and more preferably at least 95% homologousto the polypeptide of SEQ ID NO:1 over a region of at least twentycontiguous amino acids. By “fragment,” we mean peptides, oligopeptides,polypeptides, proteins and enzymes that comprise a stretch of contiguousamino acid residues, and exhibit substantially a similar, but notnecessarily identical, functional activity as the complete sequence.Fragments of SEQ ID NO:1, or their homologues, will generally be atleast ten, preferably at least fifteen, amino acids in length, and arealso encompassed by the term “a CXCL12 monomer” as used herein.

Mutations known to prevent degradation of CXCL12 or increase the in vivohalf life may also be incorporated into the CXCL12α₂ sequence. Forinstance, adding a serine to the N-terminus along with a S4Vsubstitution prevent CXCL12 degradation by proteases. Therefore, addinga serine to the N-terminus would likely similarly prevent proteasedegradation of the CXCL12α₂ locked dimer of the present invention.

Further, in addition to binding CXCR4, CXCL12 also binds to heparinfound in the extracellular matrix on cell surfaces. The inventors haveshown that the CXCL12α₂ locked dimer of the present invention also bindsheparin. Amino acid substitutions in CXCL12, including K24S, K27S, orK24S/K27S can prevent heparin binding and increase the half-life ofCXCL12 in vivo; therefore, similar mutations in CXCL12α₂ would likelyprevent heparin binding and increase the in vivo half-life of the dimer.

CXCL12α₂ variants have been generated that have a gly-met dipeptide onthe N-terminus. N-terminal extensions to CXCL12 prevent CXCR4 activationand thus inclusion in CXCL12α₂ may increase its effectiveness.Additionally, it may be useful to create CXCL12α₂ variants where bothsubunits are not identical. For example, only one monomer of theCXCL12α₂ dimer may need to include the addition of an N-terminal serineand a S4V substitution or the lysine substitutions for the prevention ofheparin binding. Alternatively, a CXCL12α₂ variant where the N-terminusof one monomer has the native sequence but the other has been extendedmay have different or enhanced pharmacological properties compared toCXCL12α₂.

The locked CXCL12 dimer could also be incorporated into a larger proteinor attached to a fusion protein that may function to increase the halflife of the dimer in vivo or be used as a mechanism for time releasedand/or local delivery (U.S. Patent Appn. No. 20060088510).

In another embodiment, the invention provides an isolated CXCL12-α₂locked dimer polypeptide as described above. By “isolated” we mean anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated. Anisolated nucleic acid is present in a form or setting that is differentfrom that in which it is found in nature. In contrast, non-isolatednucleic acids such as DNA and RNA are found in the state they exist innature. For example, a given DNA sequence (e.g., a gene) is found on thehost cell chromosome in proximity to neighboring genes; RNA sequences,such as a specific mRNA sequence encoding a specific protein, are foundin the cell as a mixture with numerous other mRNAs that encode amultitude of proteins. However, an isolated nucleic acid encoding agiven protein includes, by way of example, such nucleic acid in cellsordinarily expressing the given protein where the nucleic acid is in achromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidecan be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide can be single-stranded), but cancontain both the sense and anti-sense strands (i.e., the oligonucleotideor polynucleotide can be double-stranded).

CXCL12-α₂ locked dimer polypeptides of the present invention can beprepared by standard techniques known in the art. The peptide componentof CXCL12-α₂ is composed, at least in part, of a peptide, which can besynthesized using standard techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant, G. A. (ed.). Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). Automated peptide synthesizers arecommercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Additionally, one or more modulating groupscan be attached to the CXCL12-α₂ derived peptidic component by standardmethods, such as by using methods for reaction through an amino group(e.g., the alpha-amino group at the amino-terminus of a peptide), acarboxyl group (e.g., at the carboxy terminus of a peptide), a hydroxylgroup (e.g., on a tyrosine, serine or threonine residue) or othersuitable reactive group on an amino acid side chain (see e.g., Greene,T. W. and Wuts, P. G. M. Protective Groups in Organic Synthesis, JohnWiley and Sons, Inc., New York (1991)). Exemplary syntheses of preferredCXCL12-α₂ locked dimer polypeptides according to the present inventionare described further in the Examples below.

Peptides of the invention may be chemically synthesized using standardtechniques such as those described in Bodansky, M. Principles of PeptideSynthesis, Springer Verlag, Berlin (1993) and Grant, G. A. (ed.).Synthetic Peptides: A User's Guide, W.H. Freeman and Company, New York,(1992) (all of which are incorporated herein by reference).

In another aspect of the invention, peptides may be prepared accordingto standard recombinant DNA techniques using a nucleic acid moleculeencoding the peptide. A nucleotide sequence encoding the peptide can bedetermined using the genetic code and an oligonucleotide molecule havingthis nucleotide sequence can be synthesized by standard DNA synthesismethods (e.g., using an automated DNA synthesizer). Alternatively, a DNAmolecule encoding a peptide compound can be derived from the naturalprecursor protein gene or cDNA (e.g., using the polymerase chainreaction (PCR) and/or restriction enzyme digestion) according tostandard molecular biology techniques.

CXCL12-α₂ Locked Dimer Polypeptide Pharmaceutical Compositions. Inanother embodiment, the invention provides a composition comprising asubstantially pure CXCL12-α₂ locked dimer polypeptide of the presentinvention, and a pharmaceutically acceptable carrier. By“pharmaceutically acceptable carrier” we mean any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier may besuitable for parenteral administration. Alternatively, the carrier canbe suitable for intravenous, intraperitoneal, intramuscular, sublingualor oral administration. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, membrane nanoparticle or otherordered structure suitable to high drug concentration. The carrier canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, such as, monostearate salts and gelatin.

Moreover, the CXCL12-α₂ locked dimer polypeptide of the presentinvention can be administered in a time-release formulation, such as ina composition which includes a slow release polymer. The activecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g. CXCR4 antagonist) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The CXCL12-α₂ locked dimer polypeptide of the present invention also maybe formulated with one or more additional compounds that enhance thesolubility of the CXCL12-α₂ locked dimer polypeptide.

Administration. The CXCL12-α₂ locked dimer polypeptide of the presentinvention, optionally comprising other pharmaceutically activecompounds, can be administered to a patient orally, rectally,parenterally, (e.g., intravenously, intramuscularly, or subcutaneously)intracisternally, intravaginally, intraperitoneally, intravesically,locally (for example, powders, ointments or drops), or as a buccal ornasal spray. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

Parenteral administration of a pharmaceutical composition includes anyroute of administration characterized by physical breaching of a tissueof a human and administration of the pharmaceutical composition throughthe breach in the tissue. Parenteral administration thus includesadministration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection and intravenous,intraarterial, or kidney dialytic infusion techniques.

Compositions suitable for parenteral injection comprise the CXCL12-α₂locked dimer of the invention combined with a pharmaceuticallyacceptable carrier such as physiologically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions, or emulsions, or maycomprise sterile powders for reconstitution into sterile injectablesolutions or dispersions. Examples of suitable aqueous and nonaqueouscarriers, diluents, solvents, or vehicles include water, isotonicsaline, ethanol, polyols (e.g., propylene glycol, polyethylene glycol,glycerol, and the like), suitable mixtures thereof, triglycerides,including vegetable oils such as olive oil, or injectable organic esterssuch as ethyl oleate. Proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and/or by the use ofsurfactants. Such formulations can be prepared, packaged, or sold in aform suitable for bolus administration or for continuous administration.Injectable formulations can be prepared, packaged, or sold in unitdosage form, such as in ampules, in multi-dose containers containing apreservative, or in single-use devices for auto-injection or injectionby a medical practitioner.

Formulations for parenteral administration include suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchformulations can further comprise one or more additional ingredientsincluding suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the CXCL12-α₂locked dimer polypeptide is provided in dry (i.e., powder or granular)form for reconstitution with a suitable vehicle (e.g., sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart. Such sterile injectable formulations can be prepared using anon-toxic parenterally-acceptable diluent or solvent, such as water or1,3-butanediol, for example. Other acceptable diluents and solventsinclude Ringer's solution, isotonic sodium chloride solution, and fixedoils such as synthetic mono- or di-glycerides. Otherparentally-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

The CXCL12-α₂ locked dimer polypeptide of the present invention may alsocontain adjuvants such as suspending, preserving, wetting, emulsifying,and/or dispersing agents, including, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of injectablepharmaceutical compositions can be brought about by the use of agentscapable of delaying absorption, such as aluminum monostearate and/orgelatin.

Dosage forms can include solid or injectable implants or depots. Inpreferred embodiments, the implant comprises an effective amount of theα₂ locked dimer polypeptide and a biodegradable polymer. In preferredembodiments, a suitable biodegradable polymer can be selected from thegroup consisting of a polyaspartate, polyglutamate, poly(L-lactide), apoly(D,L-lactide), a poly(lactide-co-glycolide), a poly(c-caprolactone),a polyanhydride, a poly(beta-hydroxy butyrate), a poly(ortho ester) anda polyphosphazene. In other embodiments, the implant comprises aneffective amount of CXCL12-α₂ locked dimer polypeptide and a silasticpolymer. The implant provides the release of an effective amount ofCXCL12-α₂ locked dimer polypeptide for an extended period ranging fromabout one week to several years.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the CXCL12-α₂ lockeddimer polypeptide is admixed with at least one inert customary excipient(or carrier) such as sodium citrate or dicalcium phosphate or (a)fillers or extenders, as for example, starches, lactose, sucrose,mannitol, or silicic acid; (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, or acacia; (c) humectants, as for example, glycerol; (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, orsodium carbonate; (e) solution retarders, as for example, paraffin; (f)absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, as for example, cetyl alcohol or glycerolmonostearate; (h) adsorbents, as for example, kaolin or bentonite;and/or (i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules and tablets, the dosage forms may alsocomprise buffering agents.

A tablet comprising the active ingredient can, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture.

Tablets may be manufactured with pharmaceutically acceptable excipientssuch as inert diluents, granulating and disintegrating agents, bindingagents, and lubricating agents. Known dispersing agents include potatostarch and sodium starch glycolate. Known surface active agents includesodium lauryl sulfate. Known diluents include calcium carbonate, sodiumcarbonate, lactose, microcrystalline cellulose, calcium phosphate,calcium hydrogen phosphate, and sodium phosphate. Known granulating anddisintegrating agents include corn starch and alginic acid. Knownbinding agents include gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include magnesium stearate, stearic acid, silica, andtalc.

Tablets can be non-coated or coated using known methods to achievedelayed disintegration in the gastrointestinal tract of a human, therebyproviding sustained release and absorption of the active ingredient. Byway of example, a material such as glyceryl monostearate or glyceryldistearate can be used to coat tablets. Further by way of example,tablets can be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets. Tablets can further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Solid dosage forms such as tablets, dragees, capsules, and granules canbe prepared with coatings or shells, such as enteric coatings and otherswell known in the art. They may also contain opacifying agents, and canalso be of such composition that they release the active compound orcompounds in a delayed manner. Examples of embedding compositions thatcan be used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike. Hard capsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and can further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin. Soft gelatincapsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which can be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Dose Requirements. In particular embodiments, a preferred range fortherapeutically or prophylactically effective amounts of CXCL12-α₂locked dimer polypeptide may be 0.1 nM-0.1M, particularly 0.1 nM-0.05M,more particularly 0.05 nM-15 μM and most particularly 0.01 nM-10 μM. Itis to be noted that dosage values may vary with the severity of thecondition to be alleviated, especially with multiple sclerosis. It is tobe further understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

The amount of CXCL12-α₂ locked dimer polypeptide in the composition mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare dictated by and directly dependent on (a) the unique characteristicsof the active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch as active compound for the treatment of sensitivity in individuals.

Methods of Use. The invention also provides corresponding methods ofuse, including methods of medical treatment, in which a therapeuticallyeffective dose of a CXCL12-α₂ locked dimer polypeptide, preferablywherein the dimer comprises at least one monomer having the amino acidsequence according to SEQ ID NO:1, is administered in apharmacologically acceptable formulation. Accordingly, the inventionalso provides therapeutic compositions comprising a CXCL12-α₂ lockeddimer polypeptide and a pharmacologically acceptable excipient orcarrier, as described above. The therapeutic composition mayadvantageously be soluble in an aqueous solution at a physiologicallyacceptable pH.

In one embodiment, the invention provides a method of treatingautoimmune disease in a subject comprising administering to the subjecta therapeutically effective amount of a composition comprising aCXCL12-α₂ locked dimer polypeptide. By “autoimmune disease” we meanillnesses generally understood to be caused by the over-production ofcytokines, lymphotoxins and antibodies by white blood cells, includingin particular T-cells. Such autoimmune diseases include but are notlimited to Multiple Sclerosis (MS), Guillain-Barre Syndrome, AmyotrophicLateral Sclerosis, Parkinson's disease, Alzheimer's disease, DiabetesType I, gout, lupus, and any other human illness that T-cells play amajor role in, such as tissue graft rejection. In addition, diseasesinvolving the degradation of extra-cellular matrix include, but are notlimited to, psoriatic arthritis, juvenile arthritis, early arthritis,reactive arthritis, osteoarthritis, ankylosing spondylitis.osteoporosis, muscular skeletal diseases like tendonitis and periodontaldisease, cancer metastasis, airway diseases (COPD, asthma or otherreactive airways disease), renal and liver fibrosis, cardio-vasculardiseases like atherosclerosis and heart failure, and neurologicaldiseases like neuroinflammation and multiple sclerosis. Diseasesinvolving primarily joint degeneration include, but are not limited to,rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, earlyarthritis, reactive arthritis, osteoarthritis, ankylosing spondylitis.Diseases involving the eye include, but are not limited to autoimmuneuveitis and uveoconjunctivitis and dry eye syndrome. Diseases involvingpost-infections complications of viral or bacterial diseases such asglomerulonephritis, vasculitis, meningoencephalitis. Diseases involvingthe gastrointestinal system include but are not limited to inflammatorybowel diseases.

By “subject” we mean mammals and non-mammals. “Mammals” means any memberof the class Mammalia including, but not limited to, humans, non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, and swine; domesticanimals such as rabbits, dogs, and cats; laboratory animals includingrodents, such as rats, mice, and guinea pigs; and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.The term “subject” does not denote a particular age or sex.

By “treating” we mean the management and care of a subject for thepurpose of combating the disease, condition, or disorder. The termsembrace both preventative, i.e., prophylactic, and palliativetreatments. Treating includes the administration of a compound of thepresent invention to prevent, ameliorate and/or improve the onset of thesymptoms or complications, alleviating the symptoms or complications, oreliminating the disease, condition, or disorder.

By “ameliorate”, “amelioration”, “improvement” or the like we mean adetectable improvement or a detectable change consistent withimprovement occurs in a subject or in at least a minority of subjects,e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range about between anytwo of these values. Such improvement or change may be observed intreated subjects as compared to subjects not treated with the lockeddimer of the present invention, where the untreated subjects have, orare subject to developing, the same or similar disease, condition,symptom or the like. Amelioration of a disease, condition, symptom orassay parameter may be determined subjectively or objectively, e.g.,self assessment by a subject(s), by a clinician's assessment or byconducting an appropriate assay or measurement, including, e.g., aquality of life assessment, a slowed progression of a disease(s) orcondition(s), a reduced severity of a disease(s) or condition(s), or asuitable assay(s) for the level or activity(ies) of a biomolecule(s),cell(s) or by detection of cell migration within a subject. Ameliorationmay be transient, prolonged or permanent or it may be variable atrelevant times during or after the locked dimer of the present inventionis administered to a subject or is used in an assay or other methoddescribed herein or a cited reference, e.g., within about 1 hour of theadministration or use of the locked dimer of the present invention toabout 3, 6, 9 months or more after a subject(s) has received the lockeddimer of the present invention.

By “modulation” of, e.g., a symptom, level or biological activity of amolecule, replication of a pathogen, cellular response, cellularactivity or the like means that the cell level or activity is detectablyincreased or decreased. Such increase or decrease may be observed intreated subjects as compared to subjects not treated with the lockeddimer of the present invention, where the untreated subjects have, orare subject to developing, the same or similar disease, condition,symptom or the like. Such increases or decreases may be at least about2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more orabout within any range about between any two of these values. Modulationmay be determined subjectively or objectively, e.g., by the subject'sself assessment, by a clinician's assessment or by conducting anappropriate assay or measurement, including, e.g., quality of lifeassessments or suitable assays for the level or activity of molecules,cells or cell migration within a subject. Modulation may be transient,prolonged or permanent or it may be variable at relevant times during orafter the locked dimer of the present invention is administered to asubject or is used in an assay or other method described herein or acited reference, e.g., within about 1 hour of the administration or useof the locked dimer of the present invention to about 3, 6, 9 months ormore after a subject(s) has received the locked dimer of the presentinvention.

By “administering” we mean any means for introducing the CXCL12-α₂locked dimer polypeptide of the present invention into the body,preferably into the systemic circulation. Examples include but are notlimited to oral, buccal, sublingual, pulmonary, transdermal,transmucosal, as well as subcutaneous, intraperitoneal, intravenous, andintramuscular injection.

By “therapeutically effective amount” we mean an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as reduction or reversal of angiogenesis in thecase of cancers, or reduction or inhibition of T-cells in autoimmunediseases. A therapeutically effective amount of the CXCL12-α₂ lockeddimer polypeptide may vary according to factors such as the diseasestate, age, sex, and weight of the subject, and the ability of theCXCL12-α₂ locked dimer polypeptide to elicit a desired response in thesubject. Dosage regimens may be adjusted to provide the optimumtherapeutic response. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the CXCL12-α₂ locked dimerpolypeptide are outweighed by the therapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing or inhibiting the rate ofmetastasis of a tumor or the onset of bouts or episodes of multiplesclerosis. A prophylactically effective amount can be determined asdescribed above for the therapeutically effective amount. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

In another embodiment, the invention provides a method of treating atumor in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide. By “tumor” we mean any abnormal proliferationof tissues, including solid and non-solid tumors. For instance, thecomposition and methods of the present invention can be utilized totreat cancers that manifest solid tumors such as breast cancer, coloncancer, lung cancer, thyroid cancer, ovarian cancer and the like. Thecomposition and methods of the present invention can also be utilized totreat non-solid tumor cancers such as non-Hodgkin's lymphoma, leukemiaand the like.

In another embodiment, the present invention provides a method ofinhibiting angiogenesis in a subject by administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide. By “angiogenesis” we mean the process wherebynew blood vessels penetrate tissue thus supplying oxygen and nutrientswhile removing waste in various pathological conditions including butnot limited to diabetic retinopathy, macular degeneration, rheumatoidarthritis, inflammatory bowel disease, cancer, psoriasis,osteoarthritis, ulcerative colitis, Crohn's disease and coronarythrombosis.

In another embodiment, the present invention provides a method oftreating inflammation in a subject by administering to the subject atherapeutically effective amount of a composition comprising a CXCL12-α₂locked dimer polypeptide. By “inflammation” we mean the complexbiological response of vascular tissues to harmful stimuli, such aspathogens, damaged cells, or irritants. It is a protective attempt bythe organism to remove the injurious stimuli as well as initiate thehealing process for the tissue. For instance, the composition andmethods of the present invention can be utilized to treat inflammationassociated with: an allergic disease such as asthma, hives, urticaria,pollen allergy, dust mite allergy, venom allergy, cosmetics allergy,latex allergy, chemical allergy, drug allergy, insect bite allergy,animal dander allergy, stinging plant allergy, poison ivy allergy andfood allergy; a neurodegenerative disease; a cardiovascular disease; agastrointestinal disease; a tumor such as a malignant tumor, a benigntumor, a solid tumor, a metastatic tumor and a non-solid tumor; septicshock; anaphylactic shock; toxic shock syndrome; cachexia; necrosis;gangrene; a prosthetic implant such as a breast implant, a siliconeimplant, a dental implant, a penile implant, a cardiac implant, anartificial joint, a bone fracture repair device, a bone replacementimplant, a drug delivery implant, a catheter, a pacemaker and arespirator tube; menstruation; an ulcer such as a skin ulcer, a bedsore, a gastric ulcer, a peptic ulcer, a buccal ulcer, a nasopharyngealulcer, an esophageal ulcer, a duodenal ulcer and a gastrointestinalulcer; an injury such as an abrasion, a bruise, a cut, a puncture wound,a laceration, an impact wound, a concussion, a contusion, a thermalburn, frostbite, a chemical burn, a sunburn, a desiccation, a radiationburn, a radioactivity burn, smoke inhalation, a torn muscle, a pulledmuscle, a torn tendon, a pulled tendon, a pulled ligament, a tornligament, a hyperextension, a torn cartilage, a bone fracture, a pinchednerve and a gunshot wound; a musculo-skeletal inflammation such as amuscle inflammation, myositis, a tendon inflammation, tendinitis, aligament inflammation, a cartilage inflammation, a joint inflammation, asynovial inflammation, carpal tunnel syndrome and a bone inflammation.

In another embodiment, the locked dimer of the present invention mayalso provide for the down regulation of cell surface expression of CXCR4without activating the chemotaxis machinery of said cells. In anotherembodiment, the locked dimer of the present invention may enhance theefficacy of CXCR4 receptor pharmacological antagonists or HIV-1 entryblockers by decreasing the cell surface expression of CXCR4.

Kits. In another embodiment, the present invention provides a kitcomprising a pharmaceutical composition according to the presentinvention and instructional material. By “instructional material” wemean a publication, a recording, a diagram, or any other medium ofexpression which is used to communicate the usefulness of thepharmaceutical composition of the invention for one of the purposes setforth herein in a human. The instructional material can also, forexample, describe an appropriate dose of the pharmaceutical compositionof the invention. The instructional material of the kit of the inventioncan, for example, be affixed to a container which contains apharmaceutical composition of the invention or be shipped together witha container which contains the pharmaceutical composition.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thepharmaceutical composition be used cooperatively by the recipient.

The invention may also further comprise a delivery device for deliveringthe composition to a subject. By way of example, the delivery device canbe a squeezable spray bottle, a metered-dose spray bottle, an aerosolspray device, an atomizer, a dry powder delivery device, aself-propelling solvent/powder-dispensing device, a syringe, a needle, atampon, or a dosage-measuring container. It may be desirable to providea memory aid on the kit, e.g., in the form of numbers next to thetablets or capsules whereby the numbers correspond with the days of theregimen that the tablets or capsules so specified should be ingested.Another example of such a memory aid is a calendar printed on the card,e.g., as follows “First Week, Monday, Tuesday, . . . etc. . . . SecondWeek, Monday, Tuesday,” etc. Other variations of memory aids will bereadily apparent. A “daily dose” can be a single tablet or capsule orseveral pills or capsules to be taken on a given day.

The delivery device may comprise a dispenser designed to dispense thedaily doses one at a time in the order of their intended use isprovided. Preferably, the dispenser is equipped with a memory aid, so asto further facilitate compliance with the dosage regimen. An example ofsuch a memory aid is a mechanical counter, which indicates the number ofdaily doses that have been dispensed. Another example of such a memoryaid is a battery-powered micro-chip memory coupled with a liquid crystalreadout, or audible reminder signal which, for example, reads out thedate that the last daily dose has been taken and/or reminds one when thenext dose is to be taken.

III. Examples

The following examples describing materials and methodology are offeredfor illustrative purposes only, and are not intended to limit the scopeof the present invention.

Detection of 2:1 p38: CXCL12-α₂ locked dimer polypeptide binding.Binding the wild-type CXCL12-α with p38 requires navigating a complexset of coupled binding equilibria to permit exchange between complexeswith stoichiometries of 1:1, 1:2, 2:1 and 2:2. Because only one set ofCXCL12-α₂ locked dimer signals is observed during the p38 titration andaddition of more than two molar equivalents of p38 induces no furtherchemical shift perturbations, the inventors concluded that a symmetric2:1 p38: CXCL12-α₂ locked dimer complex is formed (see Example 3).

Structure analysis of CXCL12-α₂ locked dimer polypeptide individualsulfotyrosine recognition sites. Structures of sulfotyrosine-containingprotein complexes show that the sulfonate group typically interact witha positively charged side chain. Each negatively-charged sulfotyrosineside chains docks into a unique positively-charged pocket on theCXCL12-α₂ surface (FIG. 4), and the inventors designed a series of aminoacid substitutions in wild-type CXCL12-α to individually disrupt thosebinding sites (see Example 4 and Table 5).

Structure of CXCL12-α₂ locked dimer polypeptide bound to sulfated p38peptides. CXCR4 stabilizes the CXCL12-α₂ dimer by interacting with bothsubunits and recognizing unique features of the CXCL12-α₂ dimerinterface. Near the CXCR4 N-terminus, each p38 peptide crosses theCXCL12-α₂ dimer interface, such that sY7 and sY12 interact with opposingCXCL12-α monomers. In the membrane-proximal portion of the CXCR4N-terminal domain, P27 inserts between Q59 of one CXCL12-α₂ subunit andL66 of the opposing subunit, where the C-terminal helices from eachmonomer pack against each other (see Example 5).

Inhibitory Effect of the CXCL12-α₂ locked dimer polypeptide on cellmigration. The inventors data shows that the CXCL12-α₂ locked dimerinhibits chemotaxis induced by wild-type CXCL12-α. The CXCL12-α₂ lockeddimer inhibits chemotaxis induced by wild-type CXCL12-α with an IC₅₀ ofapproximately 4 nM (FIG. 5D). The inventors' results show that monomerCXCL12-α activates cell migration while the dimeric CXCL12-α₂ halts cellmigration. The results also demonstrate that the CXCL12-α₂ locked dimeracts as a partial CXCR4 agonist (as evidenced by the detection of thesecondary messenger calcium) and as a selective antagonist that blockschemotaxis. This indicates that the CXCL12-α₂ locked dimer is a tool forcorrelating cellular responses with different intracellular signalingpathways initiated by CXCR4. Also, the CXCL12-α₂ locked dimer of thepresent invention may be useful in the development of anti-metastaticagents by preventing CXCL12-α/CXCR4-mediated migration of circulatingcancer cells (see Example 6).

Physiological relevance of the CXCL12-α2 locked dimer. Despite beingobtained with a constitutively dimeric chemokine, CXCL12-α₂:p38structures correctly identify key elements of CXCR4 recognition bywild-type CXCL12-α. The inventors monitored activation of CXCR4 usingTHP-1 cells and a similar Ca²⁺-flux assay. Robust CXCR4 activation wasobserved with both wild-type CXCL12-α (EC₅₀=3.6 nM) and CXCL12-α₂(EC₅₀=12.9 nM) (FIG. 5A). AMD3100, a small-molecule CXCR4 antagonist,inhibited both proteins with IC₅₀ values of 3.3 nM (CXCL12-A) and 3.2 nM(CXCL12-α₂), demonstrating that the observed calcium flux responses weremediated by CXCR4. Thus, the inventors data shows that CXCL12-α₂ bindsand activates its cognate receptor (see Example 7).

Chemotaxis of the CXCL12-α₂ locked dimer. Results from Ca²⁺-flux assayscollectively suggest that CC and CXC chemokine dimers behavedifferently. For instance, CC chemokines form dimers that cannotactivate their cognate GPCRs, while CXC chemokine dimers arefunctionally indistinguishable from their monomeric counterparts. Incontrast to wild-type CXCL12-α, the constitutively dimeric CXCL12-α₂ ofthe present invention failed to attract cells in a transwell chemotaxisassay even at concentrations up to 1 μM (FIG. 5B). Accordingly, theinventors results demonstrate that at low chemokine concentrationsmonomeric CXCL12-α stimulates chemotaxis, while at higher concentrationsdimeric CXCL12-α₂ halts cell migration corresponding to the second,downward half of the bell-shaped curve (see Example 8).

The activation of CXCR4 in the calcium flux assay by CXCL12-α₂ and theinhibition of chemotaxis brings up the question of CXCL12-α₂ CXCR4stoichiometry. CXCR4 has been purified as a homodimer, the CXCR4N-terminus promotes CXCL12-α dimer formation and the inventors'structures show two CXCR4 N-termini bound to CXCL12-α₂. Nevertheless,the role of homo- and heterodimers in GPCR signaling remainscontroversial. Some place a high significance on the role of dimerformation in signaling, while others discount evidence of dimerizationas an experimental artifact.

Inspection of the high resolution, dimeric crystal structure of β₂AR, atype A GPCR like CXCR4 suggests that formation of a 2:2 CXCL12-α:CXCR4complex is plausible. The ligand binding sites of β₂ adrenergic receptormonomers are separated by approximately 42 Å, which is the distancebetween the N-termini in a CXCL12-α₂ dimer. The N-terminus of CXCL12-αactivates the CXCR4 receptor and thus is the region of CXCL12-α thatcorresponds to small molecule agonists of GPCRs like β₂AR.

Additionally, it is reasonable to propose that wild-type CXCL12concentrations near the IC₅₀ for chemotaxis prevention arephysiologically plausible. Here the inventors show that the lockedCXCL12-α₂ dimer of the present invention can inhibit chemotaxis with anIC₅₀ of 4 nM. In the presence of the CXCR4 N-terminus the CXCL12-α dimerdissociation K_(d) is 49 μM, which equates to a 4 nM dimer CXCL12-α₂concentration at a total CXCL12-α concentration of 300-400 nM. Ifinteractions with the full-length receptor or glycosaminoglycans furtherenhance CXCL12-α self-association to yield a K_(d) of 1 CXCL12-α₂ dimerconcentrations will approach 4 nM when total CXCL12-α is 50 nM, aconcentration within the physiological range.

The structure of CXCL12-α with CXCR4 provides important knowledge on therole of sulfotyrosine recognition by chemokines. The inventors' resultsalso continue to highlight the need to address the role of chemokineoligomers in chemotaxis and show the importance of investigating eachchemokine on an individual basis, since the CXCL12-α₂ dimer can bind itsreceptor while MIP-1β dimer cannot.

Additionally, both wild-type, preferentially monomeric CXCL12-α H25R anddimeric CXCL12-α₂ activate CXCR4, generating the secondary messengercalcium. However, only wild-type CXCL12-α and CXCL12-α H25R can producechemotaxis. This suggests the oligomeric state of CXCL12-α controlscertain intracellular signals that either lead to or prevent chemotaxis.Identification of the specific signaling pathways affected by dimericCXCL12-α₂ will improve understanding of cell migration and may suggestintracellular targets for the prevention of cancer metastasis.

The dimeric CXCL12 blocks the normal agonistic activity of CXCL12 butdoes not necessarily prevent the internalization or so-calleddown-regulation of the target CXCR4 receptor. Hence, CXCL12α₂ lockeddimer not only blocks the chemotactic effect of CXCL12 but also theeffective concentration of the CXCR4 receptor on the cell surfacedecreases. Thus, CXCL12α₂ locked dimer should be expected to display ahigh degree of efficacy, even when compared to standard CXCR4antagonists, which may block agonist activity, but fail to decreasereceptor number.

Example 1 Materials, Methods and Instrumentation

The production of sY₁ p38 has been explained and Y₃ p38 was generated ina similar manner. Samples for structure elucidation consisted of U-[¹⁵N,¹³C] CXCL12-α₂ with unlabeled peptide and U-[¹⁵N, ¹³C] peptide withunlabeled CXCL12-α₂ at a 1:1.25 molar ratio of labeled to unlabeledmonomers. Standard NMR techniques were use for generating chemical shiftassignments for ¹⁵N/¹³C labeled CXCL12-α₂, p38, sY₁ p38 and sY₁ p38. 3D¹⁵N-edited NOESY-HSQC, ¹³C-edited NOESY-HSQC, and ¹³C(aromatic)-editedNOESY-HSQC spectra (τ_(mix)=80 ms) were used to generate distanceconstraints. A 3D F1-¹³C-filtered/F3-¹³C-edited NOESY-HSQC spectrum(τ_(mix)=120 ms) was used for obtaining intermolecular distanceconstraints.

TALOS and the secondary shifts of the ¹H^(α), ¹³C^(α), ¹³C^(β), ¹³C′,and ¹⁵N nuclei generated backbone phi and psi dihedral angleconstraints. The NOEASSIGN module of the torsion angle dynamics programCYANA with intermolecular constraints defined was used to calculate theinitial structures in an automated manner. Iterative manual refinementfollowed to eliminate constraint violations generating twenty conformerswith the lowest target function. X-PLOR was used for further refinement,in which physical force field terms and explicit water solvent moleculeswere added to the experimental constraints. Tables 1-4 list thestatistics for Procheck-NMR validation of the final twenty conformers.

TABLE 1 Statistics for the 20 CXCL12-_(α) ₂ L36C A65C conformersExperimental constraints Distance constraints Long Intra-CXCL12-αmonomer 857 Inter-CXCL12-α monomers 113 Medium [1 < (i − j) ≦ 5] 300Sequential [(i − j) = 1] 312 Intraresidue [i = j] 692 Total 2274Dihedral angle constraints (φ and ψ) 138 Average atomic R.M.S.D. to themean structure (Å) Residues Backbone (C^(α), C′, N) 0.51 ± 0.05 Heavyatoms 1.05 ± 0.12 Deviations from idealized covalent geometry ^(a) Bondlengths RMSD (Å) 0.017 Torsion angle violations RMSD (°) 1.4 WHATCHECKquality indicators Z-score −1.52 ± 0.23   RMS Z-score Bond lengths 0.78± 0.02 Bond angles 0.76 ± 0.02 Bumps 0 ± 0 Lennard-Jones energy ^(b) (kJmol⁻¹) −2927.8 ± 108.0    Constraint violations ^(c, d) NOE distanceNumber > 0.5 Å 0 ± 0 NOE distance RMSD (Å) 0.0251 ± 0.0013 Torsion angleviolations Number > 5° 0.05 ± 0.22 Torsion angle violations RMSD (°)0.8273 ± 0.1190 Ramachandran statistics (% of all residues) Most favored81.74 ± 2.59  Additionally allowed 14.91 ± 3.05  Generously allowed 1.76± 1.03 Disallowed 1.56 ± 1.23 ^(a) Final X-PLOR force constants were 250(bonds), 250 (angles), 300 (impropers), 100 (chirality) and 100 (omega),50 (NOE constraints), and 200 (torsion angle constraints). ^(b)Nonbonded energy was calculated in XPLOR-NIH. ^(c) The largest NOEviolation in the ensemble of structures was 0.355 Å. ^(d) The largesttorsion angle violations in the ensemble of structures was 3.9°.

TABLE 2 Statistics for the 20 CXCL12-_(α) ₂ L36C A65C with CXCR4 P38C28A conformers Experimental constraints Distance constraints LongIntra-subunit 444 Inter-CXCL12-α monomers 110 Intermolecular (CXCL12-αto peptide) 92 Medium [1 < (i − j) ≦ 5] 238 Sequential [(i − j) = 1] 384Intraresidue [i = j] 744 Total 2012 Dihedral angle constraints (φ and ψ)128 Average atomic R.M.S.D. to the mean structure (Å) Residues ^(a)Chemokine Backbone (C^(α), C′, N) 0.71 ± 0.07 Chemokine Heavy atoms 1.24± 0.10 Total Backbone (C^(α), C′, N) 1.80 ± 0.24 Total Heavy atoms 2.26± 0.20 Deviations from idealized covalent geometry ^(b) Bond lengthsRMSD (Å) 0.017 Torsion angle violations RMSD (°) 1.5 WHATCHECK qualityindicators Z-score −3.47 ± 0.32   RMS Z-score Bond lengths 0.81 ± 0.03Bond angles 0.79 ± 0.03 Bumps 0 ± 0 Lennard-Jones energy ^(c) (kJ mol⁻¹)−4620.1 ± 142.7    Constraint violations ^(d) NOE distance Number > 0.5Å 0 ± 0 NOE distance RMSD (Å) 0.0276 ± 0.0017 Torsion angle violationsNumber > 5°  0.1 ± 0.45 Torsion angle violations RMSD (°) 0.8400 ±0.1693 Ramachandran statistics (% of all residues) Most favored 71.30 ±3.37  Additionally allowed 23.25 ± 3.08  Generously allowed 3.10 ± 1.09Disallowed 2.38 ± 1.34 ^(a) 20 structure in the ensemble were alignedusing residues 9-43 and 47-66 of the chemokine and 11-27 or the peptide.Chemokine RMSD includes residues 9-43, 47-66. Total RMSD includes thechemokine residues plus peptide residues 11-27. ^(b) Final X-PLOR forceconstants were 250 (bonds), 250 (angles), 300 (impropers), 100(chirality) and 100 (omega), 50 (NOE constraints), and 200 (torsionangle constraints). ^(c) Nonbonded energy was calculated in XPLOR-NIH.^(d) The largest NOE violation in the ensemble of structures is Å.

TABLE 3 Statistics for the 20 CXCL12-_(α) ₂ L36C A65C with sY21 CXCR4P38 C28A conformers Experimental constraints Distance constraints LongIntra-subunit 418 Inter-CXCL12-α monomers 116 Intermolecular (CXCL12-αto peptide) 92 Medium [1 < (i − j) ≦ 5] 246 Sequential [(i − j) = 1] 470Intraresidue [i = j] 728 Total 2070 Dihedral angle constraints (φ and ψ)128 Average atomic R.M.S.D. to the mean structure (Å) Residues ^(a)Chemokine Backbone (C^(α), C′, N) 0.60 ± 0.10 Chemokine Heavy atoms 1.05± 0.11 Total Backbone (C^(α), C′, N) 0.82 ± 0.09 Total Heavy atoms 1.37± 0.10 Deviations from idealized covalent geometry ^(b) Bond lengthsRMSD (Å) 0.018 Torsion angle violations RMSD (°) 1.6 WHATCHECK qualityindicators Z-score −3.34 ± 0.21   RMS Z-score Bond lengths 0.83 ± 0.02Bond angles 0.83 ± 0.03 Bumps 0 ± 0 Lennard-Jones energy ^(c) (kJ mol⁻¹)−4640.2 ± 199.2    Constraint violations ^(d) NOE distance Number > 0.5Å 0 ± 0 NOE distance RMSD (Å) 0.0324 ± 0.0016 Torsion angle violationsNumber > 5° 0.0 ± 0.0 Torsion angle violations RMSD (°) 0.7907 ± 0.1300Ramachandran statistics (% of all residues) Most favored 70.21 ± 2.72 Additionally allowed 24.29 ± 2.80  Generously allowed 3.71 ± 1.18Disallowed 1.74 ± 1.02 ^(a) 20 structure in the ensemble were alignedusing residues 9-43 and 47-66 of the chemokine and 11-27 or the peptide.Chemokine RMSD includes residues 9-43, 47-66. Total RMSD includes thechemokine residues plus peptide residues 11-27. ^(b) Final X-PLOR forceconstants were 250 (bonds), 250 (angles), 300 (impropers), 100(chirality) and 100 (omega), 50 (NOE constraints), and 200 (torsionangle constraints). ^(c) Nonbonded energy was calculated in XPLOR-NIH.^(d) The largest NOE violation in the ensemble of structures was 0.47 Å.

TABLE 4 Statistics for the 20 CXCL12-_(α) ₂ L36C A65C with sY7-12-21CXCR4 P38 C28A conformers. Experimental constraints Distance constraintsLong Intra-subunit 420 Inter-CXCL12-α monomers 116 Intermolecular(CXCL12-α to peptide) 86 Medium [1 < (i − j) ≦ 5] 238 Sequential [(i −j) = 1] 456 Intraresidue [i = j] 722 Total 2092 Dihedral angleconstraints (φ and ψ) 128 Average atomic R.M.S.D. to the mean structure(Å) Residues ^(a) Chemokine Backbone (C^(α), C′, N) 0.64 ± 0.07Chemokine Heavy atoms 1.10 ± 0.09 Total Backbone (C^(α), C′, N) 1.01 ±0.16 Total Heavy atoms  1.56 ± 0.017 Deviations from idealized covalentgeometry ^(b) Bond lengths RMSD (Å) 0.017 Torsion angle violations RMSD(°) 1.5 WHATCHECK quality indicators Z-score −3.60 ± 0.25   RMS Z-scoreBond lengths 0.78 ± 0.02 Bond angles 0.80 ± 0.02 Bumps 0 ± 0Lennard-Jones energy ^(c) (kJ mol⁻¹) −4766.8 ± 145.4    Constraintviolations ^(d) NOE distance Number > 0.5 Å 0 ± 0 NOE distance RMSD (Å)0.0260 ± 0.0017 Torsion angle violations Number > 5° 0 ± 0 Torsion angleviolations RMSD (°) 0.7380 ± 0.1314 Ramachandran statistics (% of allresidues) Most favored 74.03 ± 2.65  Additionally allowed 22.13 ± 2.70 Generously allowed 2.15 ± 1.01 Disallowed 1.72 ± 1.22 ^(a) 20 structurein the ensemble were aligned using residues 9-43 and 47-66 of thechemokine and 11-27 or the peptide. Chemokine RMSD includes residues9-43, 47-66. Total RMSD includes the chemokine residues plus peptideresidues 11-27. ^(b) Final X-PLOR force constants were 250 (bonds), 250(angles), 300 (impropers), 100 (chirality) and 100 (omega), 50 (NOEconstraints), and 200 (torsion angle constraints). ^(c) Nonbonded energywas calculated in XPLOR-NIH. d The largest NOE violation in the ensembleof structures was 0.407 Å.

The Protein Data Bank (PDB), under accession numbers 2K01, 2K04, 2K03and 2K05 contain coordinates for these structural models. Restraintsemployed for structure determination have been deposited in theBiological Magnetic Resonance Bank, accession numbers 15633, 15636,15635 and 15637. Standard calcium flux assays were used for testingCXCR4 activation and transwell chemotaxis assays were used to comparethe chemotactic response of THP-1 cells towards wild-type CXCL12-α,CXCL12-α H25R and CXCL12-α₂. THP-1 cells are a CXCR4-expressing monocyteleukemia cell line and were obtained from ATCC.

Example 2 Preparing the CXCL12-α₂ Locked Dimer Polypeptide

In this Example, the inventors prepared the CXCL12-α₂ locked dimerstructure. Guided by the CXCL12-α crystal structure, the inventorsidentified L36 and A65 as residues at the dimer interface that could bereplaced with intermolecular disulfide bonds (FIG. 1A). The CXCL12-α(L36C/A65C) double mutant was expressed and purified from E. coli aspreviously described for wild-type CXCL12-α, and migrated as a stabledimer in non-reducing SDS-PAGE (FIG. 1B). Pulsed-field gradient NMRdiffusion measurements indicated that CXCL12-α (L36C/A65C) is dimeric,even in solution conditions that favor the monomeric state (FIG. 1C).

The inventors confirmed the presence of disulfide bonds linking the twomonomers and solved the structure of the CXCL12-α₂ (L36C/A65C) by NMR.The covalently-locked, symmetric CXCL12-α (L36C/A65C) dimer (CXCL12-α₂)is superimposable with the wild-type CXCL12-α dimer observedcrystallographically (FIG. 2A). Table 1 lists refinement statistics forthe CXCL12-α₂ structure ensemble. CXCL12-α₂ also displays the canonicalchemokine fold in which a flexible N-terminus is connected by the N-loopto a three-stranded antiparallel β-sheet and a C-terminal α-helix.

Example 3 Detection of 2:1 p38: CXCL12-α₂ Binding by NMR Chemical ShiftMapping

In this Example, the inventors determined if the NMR broadening arisesfrom exchange between different CXCL12-α:p38 complexes. Previously, theinventors noted that binding of p38 (FIG. 5D) to ¹⁵N labeled CXCL12-αinduced chemical shift perturbations attributable to a combination ofCXCL12-α dimer formation and peptide binding. Titration of ¹⁵N p38 withCXCL12-α showed extreme line broadening. Based on the inventors' studiesof the CXCL12-α monomer-dimer equilibrium, the inventors investigatedthat the NMR broadening arises from exchange between differentCXCL12-α:p38 complexes.

When both CXCL12-α dimerization and p38 binding are considered, acomplex set of coupled binding equilibria permits exchange betweencomplexes with stoichiometries of 1:1, 1:2, 2:1 and 2:2. Because thelocked dimer reduces the number of accessible states, interpretation ofNMR spectra of CXCL12-α₂ upon p38 binding is straightforward. Titrationof ¹⁵N labeled CXCL12-α₂ with p38 (FIG. 2B) perturbs NMR signals forN-loop residues but not the dimer interface (FIG. 2C), thus identifyinglikely CXCR4-CXCL12-α binding determinants. Because only one set ofCXCL12-α₂ signals is observed during the p38 titration and addition ofmore than two molar equivalents of p38 induces no further chemical shiftperturbations, the inventors concluded that a symmetric 2:1 p38:CXCL12-α₂ complex was formed.

Example 4 Structure Analysis of CXCL12-α₂ Locked Dimer SulfotyrosineRecognition Sites

In this Example, the inventors measured the EC₅₀ of each protein using aCa²⁺-flux assay on CXCR4-expressing THP-1 cells to assess the relativecontribution of each sulfotyrosine to CXCL12-α:CXCR4 binding. TheCXCL12-α:p38 interaction contributes only to binding affinity andreceptor specificity, but not to CXCR4 activation. In contrast, apeptide consisting of CXCL12-α residues 1-8 can fully activate CXCR4 atmicromolar concentrations. Since each CXCL12-α variant retains thenative N-terminus, the EC₅₀ value reflects its affinity for CXCR4.Consequently, an amino acid substitution that alters the Ca²⁺-flux EC₅₀relative to wild-type CXCL12-α (3.6±1.4 nM) has necessarily disrupted aninteraction between the chemokine and the N-terminus or extracellularloops of CXCR4. Overall, mutations in wild-type CXCL12-α that alterinteractions observed in the CXCL12-α₂:p38 complexes resulted in higherEC₅₀ values for CXCR4 activation corresponding to a loss of CXCL12-αbinding affinity (Table 5). However, comparison of the results for eachbinding site reveals a hierarchy among CXCR4 sulfotyrosines.

NOE constraints from valine 23 of one CXCL12-α₂ subunit position the sY7O-sulfonate to form a favorable electrostatic interaction with apositively-charged arginine side chain (FIG. 4B), but replacing R20 withalanine in wild-type CXCL12-α produced no change in EC₅₀ (Table 5).

TABLE 5 CXCR4 activation by CXCL12-α mutants. Fold EC₅₀ (nM) FoldedIncrease p38 contact CXCL12-α 3.6 ± 1.4 + R20A 4.3 ± 0.6 + 1.2 + V23A NA− NA + H25R 5.1 ± 0.9 + 1.4 − K27A 10.1 ± 2.9  + 2.8 + K27E 16.8 ±1.1  + 4.7 + V39A 27.1 ± 0.2  + 7.5 + R41A 4.3 ± 0.9 + 1.2 − R47A 14.1 ±0.6  + 3.9 + R47E 654 ± 93  + 181.7 + V49A 8.6 ± 2.4 + 2.4 + E60A 4.1 ±0.1 + 1.1 − E63A 3.7 ± 0.8 + 1.0 − K64A 5.0 ± 1.1 + 1.4 −

In a similar fashion, NOEs connect sY12 to P10 and L29 of the otherCXCL12-α₂ subunit and place the sulfotyrosine within approximately 3 Åof the positively charged amino group of K27 (FIG. 4C). Substitutions ofalanine and glutamic acid at this position in wild-type CXCL12-αincreased the EC₅₀ to 10.1 and 16.8 nM, respectively. Alaninesubstitution of a structurally adjacent valine residue (V39A) increasedthe EC₅₀ to 27.1 nM.

Residues connecting the N-terminal CXC motif with β1 of CXCL12-α (the ‘Nloop’), particularly the RFFESH motif consisting of residues 12-17, werepredicted from mutagenic studies to interact with the CXCR4 N-terminus.The inventors observed intermolecular NOEs between ¹H^(N) of F14 inCXCL12-α₂ and the ¹H^(α) of G19 from CXCR4 and from V18 in the chemokineto sY21. NOEs also link sY21 with V49, located in the β3 strand ofCXCL12-α₂, and position the sY21 O-sulfonate <5 Å from the R47guanidinium (FIG. 4D), consistent with our earlier measurement ofsulfotyrosine-specific chemical shift perturbations. CXCL12-α R47A hasan EC₅₀ of 14.1 nM, and replacement of the positive arginine side chainwith a negatively charged glutamic acid drastically alters CXCL12-αbinding (R47E EC₅₀=654 nM) relative to wild-type CXCL12-α (EC₅₀=3.6 nM).

The level of sulfation for each CXCR4 tyrosine has not beencharacterized in THP-1 cells, but Farzan et al. suggested that CXCR4 Y21is sulfated to higher degree than Y7 or Y12 and that sY21 contributesthe most to CXCL12-α binding affinity. This is consistent with theresults herein which suggest the sY7 and sY12 binding sites contributeonly modestly to the overall interaction. The binding pocket for sY21 inCXCL12-α appears to be well conserved within the CXC chemokine familywith 8 out of 16 CXC chemokines showing high conservation or identity atCXCL12-α positions 18, 47 and 49. With the exception of CXCR6, atyrosine corresponding to sulfotyrosine 21 of CXCR4 appear to be presentin all receptors of the CXC family. Neither sY7, sY12 nor their putativebinding sites are conserved in the CXC ligands or receptors.

Example 5 Structure of CXCL12-α₂ Locked Dimer Bound to Sulfated p38Peptides

In this Example, the inventors solved structures of unsulfated,selectively sulfated and fully sulfated CXCR4 peptides bound to theCXCL12-α₂ locked dimer polypeptide to understand the role ofsulfotyrosine in CXCL12-a-CXCR4 binding.

Tyrosine sulfation in the CXCR4 N-terminal domain contributessubstantially to CXCL12-α binding. The inventors showed previously thatsulfation of Tyr 21 enhances the affinity of p38 for CXCL12-α byapproximately 3-fold, and the inventors observed that fully-sulfatedp38-sY₃ binds approximately 20-fold more tightly than the unsulfatedpeptide (apparent K_(d)=0.2±0.2 μM).

Recombinant [U-¹⁵N, ¹³C]-labeled CXCR4 peptide (p38) was modified usingpurified tyrosyl protein sulfotransferase to contain sulfotyrosine atposition 21 (p38-sY₁) or positions 7, 12 and 21 (p38-sY₃) (FIG. 1D). Foreach complex, NOEs between CXCL12-α₂ and the (sulfo)tyrosine side chainsof CXCR4 unambiguously defined the same arrangement of both p38 peptideson the chemokine as shown in FIG. 3 with representative intermolecularNOEs in FIG. 1B.

Two p38 molecules bind in equivalent orientations with each peptidewrapping around the symmetric CXCL12-α₂ dimer in an extendedconformation (FIG. 2D). When mapped onto the CXCL12-α₂ surface,p38-induced chemical shift perturbations (FIG. 2D, green surface)correlate strongly with the observed binding interface. In contrast,residues of the flexible N-terminus and C-terminal a-helix of CXCL12-α₂were unperturbed by p38 binding and do not interact with the CXCR4N-terminus.

CXCR4 stabilizes the CXCL12-α dimer by interacting with both subunitsand recognizing unique features of the dimer interface. Near the CXCR4N-terminus, each p38 peptide crosses the CXCL12-α₂ dimer interface, suchthat sY7 and sY12 interact with opposing CXCL12-α monomers. In themembrane-proximal portion of the CXCR4 N-terminal domain, P27 insertsbetween Q59 of one CXCL12-α₂ subunit and L66 of the opposing subunit,where the C-terminal helices from each monomer pack against each other.

Example 6 Inhibitory Effect of CXCL12-α₂ Locked Dimer on Cell Migration

In this example, the inventors conducted chemotaxis assays using aC-CXCL12-α mutant that remains monomeric at higher concentrations thanwild-type CXCL12-α. Since the dimer K_(d) of CXCL12-α (H25R) isapproximately 10-fold higher than wild-type, it should resistinactivation due to dimerization and maintain a chemotactic response athigher concentrations where the wild-type CXCL12-α loses activity. Bothproteins induce a dose-dependent chemotactic response from 1-30 nM, butCXCL12-α(H25R) promotes cell migration much more strongly than thewild-type chemokine at higher concentrations (70-100 nM) beforereturning to baseline levels (FIG. 5C).

The inventors data shows that CXCL12-α₂ inhibits chemotaxis induced bywild-type CXCL12-α. FIG. 5D shows CXCL12-α₂ inhibits chemotaxis inducedby wild-type CXCL12-α with an IC₅₀ of approximately 4 nM. The inventors'results show that monomer CXCL12-α activates cell migration while dimerCXCL12-α halts cell migration. The results also demonstrate thatCXCL12-α₂ acts as a partial CXCR4 agonist (as evidenced by the detectionof the secondary messenger calcium) and as a selective antagonist thatblocks chemotaxis. This indicates CXCL12-α₂ can serve as a tool forcorrelating cellular responses with different intracellular signalingpathways initiated by CXCR4. Also, CXCL12-α₂ may be useful in thedevelopment of anti-metastatic agents by preventingCXCL12-α/CXCR4-mediated migration of circulating cancer cells.

Example 7 Physiological Relevance of the CXCL12-α₂ Locked Dimer

In this Example, the inventors next investigated whether CXCL12-α₂locked dimers participate in CXCR4 signaling. Like most chemokines,CXCL12-α self-association occurs well above the concentrations requiredfor receptor binding and activation (1-10 nM). Consequently, chemokinedimers are considered relevant mainly in the context ofglycosaminoglycan binding for immobilization in the extracellularmatrix. Debate over the functional role of chemokine dimers in vivo iscomplicated by conflicting results obtained on a variety of the >40different chemokine proteins.

The inventors monitored activation of CXCR4 using THP-1 cells and asimilar Ca²⁺-flux assay. Robust CXCR4 activation was observed with bothwild-type CXCL12-α (EC₅₀=3.6 nM) and CXCL12-α₂ (EC₅₀=12.9 nM) (FIG. 5A).AMD3100, a small-molecule CXCR4 antagonist, inhibited both proteins withIC₅₀ values of 3.3 nM (CXCL12-A) and 3.2 nM (CXCL12-α₂), demonstratingthat the observed calcium flux responses were mediated by CXCR4. Thus,the inventors data shows that CXCL12-α₂ binds and activates its cognatereceptor.

Example 8 Chemotactic Response of the CXCL12-α₂ Locked Dimer Polypeptide

In this Example, the inventors used standard transwell chemotaxis assaysto compare the chemotactic response of THP-1 cells towards wild-typeCXCL12-α and the CXCL12-α₂ locked dimer polypeptide of the presentinvention. As expected, the THP-1 cells responded chemotactically whenexposed to wild-type CXCL12-α in the 1-30 nM range, but migrationdecreases and ultimately ceases at higher chemokine concentrations (FIG.5B).

In contrast to wild-type CXCL12-α, the constitutively dimeric CXCL12-α₂of the present invention failed to attract cells in a transwellchemotaxis assay even at concentrations up to 1 μM (FIG. 5B).Accordingly, the inventors results demonstrate that at low chemokineconcentrations monomeric CXCL12-α stimulates chemotaxis, while at higherconcentrations CXCL12-α₂ halts cell migration corresponding to thesecond, downward half of the bell-shaped curve (see Example 8).

Example 9 Prophetic Example on the Inhibitory Effect of the CXCL12-α₂Locked Dimer on Tumor Growth

In this Example, the inventors describe how one would used theinhibitory effect of the CXCL12-α₂ locked dimer of the present inventionto affect tumor growth. Primary tumors are easier to treat as comparedto cancer which has spread through the body and formed secondary tumorsor metastases. It has long been observed that secondary tumors inmetastatic cancer patients form preferentially in a subset of tissues,including bone marrow, lymph nodes, liver, and lungs. At least threetheories have been proposed to explain these observations, including:certain tissues are better environments for metastatic cancer cellsurvival; the vasculature of some tissues express adhesion moleculesthat bind metastatic cells better than others; or there is activerecruitment of metastatic cells out of the blood stream or lymphaticsystem only to certain locations.

Patterns of CXCR4 and CXCL12 expression and signaling suggest thatmetastatic cancer cells are actively recruited to tissues producingCXCL12. An increase in CXCR4 expression accompanies the transition froma primary tumor cell to a metastatic cancer cell, and CXCR4 levels havebeen correlated with metastasis and poor patient outcomes in manydifferent cancer types. These metastatic, CXCR4-expressing cancer cellsbreak away from the primary tumor and enter the circulation where theysystematically target tissues constitutively expressing CXCL12, the onlynatural ligand for CXCR4, like bone marrow, lymph nodes, liver, andlungs. It is thought that this targeting occurs in a manner analogous tothe recruitment of a circulating leukocyte to an infection site.

CXCL12- and CXCR4-directed localization of metastatic cancer cells hasbeen implicated in a broad range of over twenty cancer types, including:breast, prostate, colon, myeloma, melanoma, tongue, ovarian, small andnon-small cell lung cancers, pancreatic, esophageal, head and neck,bladder, osteosarcoma, neuroblastoma, and leukemia.

The inventors have shown that the CXCL12-α₂ locked dimer of the presentinvention is a potent inhibitor of CXCL12/CXCR4 mediated chemotaxis of aTHP1 cells (a leukemia cell line) (FIG. 5E). This CXCL12/CXCR4 mediatedchemotaxis or cell migration is required for CXCL12/CXCR4 directedcancer metastasis. Based on the potent inhibition of CXCL12 induced THP1cell chemotaxis by CXCL12-α₂, the inventors predict that the CXCL12-α₂locked dimer of the present invention will prevent CXCL12-inducedchemotaxis (or cell migration) and thus metastasis of cancer cells thatexpress CXCR4. The inventors therefore predict that blockade of theCXCL12/CXCR4-directed metastasis with the CXCL12-α₂ locked dimer of thepresent invention will prevent or reduce the formation of secondarytumors or metastases. For example, a person diagnosed with breast cancercould be treated with CXCL12-α₂ to prevent metastasis before and aftersurgical removal of the primary tumor. Continued CXCL12-α₂administration could improve the efficacy of subsequent chemotherapy orradiation treatments by preventing circulating cancer cells frommigrating to tissues and organs that would normally serve as a preferredlocation for metastatic cancer growth. The inventors predict that fewerrecurrences or metastases would occur after a successful initialtreatment of breast cancer. Pancreatic cancer has a high mortality rateand kills quickly, largely because pancreatic cancer metastasizesrapidly in a CXCL12/CXCR4 dependant manner. In a manner analogous tobreast cancer the inventors predict that treatment with CXCL12-α₂ wouldincrease the life expectancy of a patient with pancreatic cancer byslowing the spread of metastatic disease.

Example 10 Prophetic Example on the Inhibitory Effect of the CXCL12-α₂Locked Dimer on Angiogenisis

In this example, the inventors show how one would use the inhibitoryeffect of the CXCL12-α₂ locked dimer of the present invention to inhibitangiogenesis. Angiogenesis is the formation of new blood vessels thatpenetrate a tissue and supply that tissue with oxygen and nutrientswhile also removing waste. Usually the formation of new blood vesselsresults from expansion or growth of existing blood vessels through thegrowth of vascular endothelial cells. The vascular endothelial cellsthat line blood vessels rarely divide but there are cues that can induceor inhibit growth. When the expansion cues outweigh the inhibiting cues,angiogenesis occurs.

CXCL12 expression is increased in tissues that are hypoxic (or lackoxygen) due to a lack of vascularization (or lack of blood vessels andblood supply). CXCL12 is also a chemoattractant that functions toattract the newly forming blood vessels into the hypoxic tissue.Angiogenesis is important for the progression of numerous disease statesand inhibition of angiogenesis by CXCL12α₂ may be therapeutically usefulin the prevention or progression of diseases conditions including butnot limited to diabetic retinopathy, macular degeneration, rheumatoidarthritis, inflammatory bowel disease, cancer, psoriasis,osteoarthritis, ulcerative colitis, Crohn's disease and coronarythrombosis.

Example 11 Prophetic Example on the Inhibitory Effect of the CXCL12-α₂Locked Dimer on Autoimmune Diseases

In this example, the inventors show how one would use the inhibitoryeffect of the CXCL12-α₂ locked dimer of the present invention to inhibitautoimmune diseases.

Autoimmune diseases occur when a body's immune system attacks its owncells and tissues in addition to or instead of things foreign. CXCL12 isa chemokine and like other chemokines is involved in regulating immunecell trafficking and the immune system in general. As such, CXCL12 playsroles in recruiting immune cells during an autoimmune reaction.Rheumatoid arthritis is an example of such an autoimmune disease. Inrheumatoid arthritis CXCL12 recruits T-cells to joints where therecruited T-cells orchestrate the generation of immunologically driveninflammation. Therefore, preventing the migration and recruitment ofT-cells to the synovium of joints with the CXCL12α₂ locked dimer of thepresent invention may prevent or reduce the inflammation associated withrheumatoid arthritis.

Example 12 Prophetic Example on the Inhibitory Effect of the CXCL12-α₂Locked Dimer on HIV/AIDS

In this example, the inventors show how one would use the inhibitoryeffect of the CXCL12-α₂ locked dimer of the present invention to inhibitHIV/AIDS. In this example, the inventors show how one would alsoevaluate the effect of combination therapies using the inhibitory effectof the CXCL12-α₂ locked dimer of the present invention to treatHIV/AIDS.

Human immunodeficiency virus (HIV) is a retrovirus that can lead toacquired immunodeficiency syndrome (AIDS), a condition in humans inwhich the immune system begins to fail, leading to life-threateningopportunistic infections. Infection with HIV occurs by the transfer ofcontaminated bodily fluids such as blood, semen, vaginal fluid,pre-ejaculate, or breast milk. Within these bodily fluids, HIV ispresent as both free virus particles and virus within infected immunecells. CXCR4 and/or CCR5 along with CD4 are coreceptors that HIV useswhen infecting cells. There are different strains of HIV. R5 strains useCCR5 and CD4 as coreceptors to infect macrophages. X4 strains use CXCR4and CD4 to gain entrance into T cells while dual tropic HIV strains canuse either CXCR4 or CCR5 along with CD4 as coreceptors when infectingcells.

HIV primarily infects vital cells in the human immune system such ashelper T cells (specifically CD4⁺ T cells), macrophages and dendriticcells. HIV infection leads to low levels of CD4⁺ T cells through threemain mechanisms: firstly, direct viral killing of infected cells;secondly, increased rates of apoptosis in infected cells; and thirdly,killing of infected CD4⁺ T cells by CD8 cytotoxic lymphocytes thatrecognize infected cells. When CD4⁺ T cell numbers decline below acritical level, cell-mediated immunity is lost, and the body becomesprogressively more susceptible to opportunistic infections. Ifuntreated, eventually most HIV-infected individuals develop AIDS(Acquired Immunodeficiency Syndrome) and die; however about one in tenremains healthy for many years, with no noticeable symptoms. Treatmentwith anti-retrovirals, where available, increases the life expectancy ofpeople infected with HIV.

Based on the inhibitory effect of the CXCL12-α₂ locked dimer of thepresent invention, the inventors predict that the CXCL12-α₂ locked dimerwould be effective in blocking the entry of HIV-1 into human T cells.Certain strains of HIV (X4 or dual tropic) utilize CXCR4 as aco-receptor, and this interaction is critical for fusion of the viraland T cell membranes in the process of HIV entry. By binding to CXCR4and preventing it from serving as a viral coreceptor, the wild-typeCXCL12-α chemokine inhibits HIV entry with an IC50 of 79 nM (9, 38).Accordingly, the inventors predict that the CXCL12-α₂ locked dimer ofthe present invention will be more effective at lower concentrationssince it should block the CXCR4 receptor more completely than themonomeric, wild-type chemokine. Additionally, the inventers predict theCXCL12-α₂ locked dimer will work synergistically or additively withHAART, a combination therapy, or other current HIV/AIDS treatments.

Example 13 Prophetic Example of the Inhibitory Effect of the CXCL12α₂Locked Dimer on Blood Cancers

In this example, the inventors show how one would use the inhibitoryeffect of the CXCL12-α₂ locked dimer of the present invention to treatblood cancers.

Blood cancers cells such as leukemia, lymphoma and myeloma that expressCXCR4 are trafficked throughout the body in response to CXCL12. BlockingCXCR4 signaling can help prevent this trafficking, thus exposing thesecancers to treatments, like chemotherapy, resulting in an enhancedeffectiveness of the current therapy. CXCL12 traffics these cancers tothe bone marrow, which provides a protective environment that enhancesproliferation and anti-apoptotic signals that can result in the cancersbeing less sensitive to the therapies currently used. The inventors haveshown that the CXCL12α₂ locked dimer of the present invention inhibitsmigration of THP-1 cells, a leukemia cell line, toward CXCL12 (FIG. 5).Therefore, adding the CXCL12α₂ locked dimer of the present invention tothe current treatment of blood cancers, like leukemia, lymphoma andmyeloma, will prevent the migration of these cancers into the bonemarrow in response to the CXCL12 that is produced there. Thus, thecancer cells will not enter the protective bone marrow and will be moresensitive and responsive to the current cytotoxic therapeutics.

Example 14 Prophetic Example of the Inhibitory Effect of the CXCL12α₂Locked Dimer on IBD

In this example, the inventors show how one would use the inhibitoryeffect of the CXCL12-α₂ locked dimer of the present invention to treatgastrointestinal inflammation associated with IBD.

By “gastrointestinal inflammation” we mean inflammation of a mucosallayer of the gastrointestinal tract, and encompass acute and chronicinflammatory conditions. Acute inflammation is generally characterizedby a short time of onset and infiltration or influx of neutrophils.Chronic inflammation is generally characterized by a relatively longerperiod of onset and infiltration or influx of mononuclear cells. By“chronic gastrointestinal inflammatory conditions” (also referred to as“chronic gastrointestinal inflammatory diseases”) we mean, but are notnecessarily limited to, inflammatory bowel disease (IBD), colitisinduced by environmental insults (e.g., gastrointestinal inflammation(e.g., colitis) caused by or associated with (e.g., as a side effect) atherapeutic regimen, such as administration of chemotherapy, radiationtherapy, and the like), colitis in conditions such as chronicgranulomatous disease, celiac disease, celiac sprue (a heritable diseasein which the intestinal lining is inflamed in response to the ingestionof a protein known as gluten), food allergies, gastritis, infectiousgastritis or enterocolitis (e.g., Helicobacter pylori-infected chronicactive gastritis) and other forms of gastrointestinal inflammationcaused by an infectious agent, and other like conditions.

By “inflammatory bowel disease” or “IBD” we mean any of a variety ofdiseases characterized by inflammation of all or part of the intestines.Examples of IBD include, but are not limited to, Crohn's disease andulcerative colitis. Reference to IBD throughout the specification isoften referred to in the specification as exemplary of gastrointestinalinflammatory conditions, and is not meant to be limiting.

Clinical and experimental evidence suggest that the pathogenesis of IBDis multifactorial involving susceptibility genes and environmentalfactors. The interaction of these factors with the immune system leadsto intestinal inflammation and dysregulated mucosal immunity againstcommensal bacteria, various microbial products (e.g., LPS) or antigens.Animal models of colitis have highlighted the prominent role of CD4+ Tcells in the regulation of intestinal inflammation. Cytokine imbalance,and the production of inflammatory mediators have been postulated toplay an important role in the pathogenesis of both experimental colitisand IBD. In particular, dysregulated CD4+ T cell responses play apivotal role in the pathogenesis of experimental colitis. Therefore,adding the CXCL12α₂ locked dimer of the present invention to the currenttreatment of IBD will prevent the migration of these inflammatorymediators.

The invention provides a new and potent therapeutic advantage that iseffective across species in a variety of animal models of chronic and/oracute gastrointestinal inflammation, particularly in animal models ofIBD, which animal models are regarded in the field as models of diseasein humans. In use, the composition and methods of the present inventionwill reduce disease activity, e.g., diarrhea, rectal bleeding and weightloss, reduce colon weight and colon lesions, as well as reduce colonicinflammation, as measured by, for example, anti-neutrophil cytoplasmicantibodies (ANCA), colonic myclo-peroxidase activity, or otherconventional indicator of gastrointestinal inflammation.

Example 15 Prophetic Example of the Effect of Combination TherapiesUsing the CXCL12α₂ Locked Dimer on Cancer, Inflammation, Auto-ImmuneDiseases and/or HIV/AIDS

In this example, the inventors show how one would evaluate the effect ofcombination therapies using the inhibitory effect of the CXCL12-α₂locked dimer of the present invention to treat cancer, inflammation,auto-immune diseases and/or HIV/AIDS.

For instance, the locked dimer of the present invention may be used asan agonist or antagonist in combination with other knownanti-inflammatory, HIV, autoimmune or cancer therapies. By “agonist” wemean a ligand that stimulates the receptor the ligand binds to in thebroadest sense. An “agonist” or an “antagonist” is a compound orcomposition that, respectively, either detectably increases or decreasesthe activity of a receptor, an enzyme or another biological molecule,which can lead to increased or decreased transcription or mRNA levels ofa regulated gene or to another measurable effect such as altered levelof activity of the gene product or protein. The increase or decrease ina receptor's or enzyme's activity will be an increase or a decrease ofat least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or arange about between any two of these values, for one or more measurableactivities. Receptors, their accessory factors and associatedtranscription factors can modulate transcription of their target gene(s)by detectably increasing or decreasing transcription or mRNA levels.Biological activities of receptors may also include modulatingbiological responses such as signal transduction within a cell or ionflux, e.g., sodium, potassium or calcium, across cell or organellemembranes, e.g., across mitochondria.

The locked dimer of the present invention may also be used as asuper-agonist. By “super-agonist”, we mean a type of agonist that bindspermanently to a receptor in such a manner that the receptor ispermanently activated. It is distinct from a mere agonist in that theassociation of an agonist to a recepter is reversible, whereas thebinding of an super-agonist to a receptor is, at least in theory,irreversible.

In use, the locked dimer of the present invention allows b-arrestinmediated receptor internalization and down-regulation. This is a majoradvantage of the dimer since the combination of antagonism with respectto CXCL12 induced migration AND ongoing CXCL12 dimer induced receptorinternalization is profoundly synergistic. Further, the dimer of thepresent invention may also be synergistic when used with other CXCR4pharmacophores.

A. Inflammation.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000). Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962). This assay has been used as a primary in vivo screenfor the anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group. Additionally, animal models forinflammatory bowel disease can also be used to assess the efficacy ofthe combination therapies of invention.

Animal models for asthma can also be used to assess the efficacy of thecombination therapies of invention. An example of one such model is themurine adoptive transfer model in which aeroallergen provocation of TH1or TH2 recipient mice results in TH effector cell migration to theairways and is associated with an intense neutrophilic (TH1) andeosinophilic (TH2) lung mucosal inflammatory response (Cohn et al.,1997, J. Exp. Med. 186, 1737-1747).

B. Auto-Immune Disorders.

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have beendeveloped. Further, any assays known to those skilled in the art can beused to evaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Animal models for autoimmune and/or intestinal inflammation can also beused to test the efficacy of the combination therapies of the invention.An example of one such model is the murine dextran sodium sulfatecolitis model as described in Wirtz S. et al., “Chemically induced mousemodels of intestinal inflammations” Nature Protocols 2, 541-546, (2007).

C. Cancer.

The anti-cancer activity of the therapies used in accordance with thepresent invention can also be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC 50 (i.e.,the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by a variety of methodsknown to the art, including by direct cell count, by detecting changesin transcriptional activity of known genes such as proto-oncogenes orcell cycle markers; cell viability can be assessed by trypan bluestaining, differentiation can be assessed visually based on changes inmorphology, decreased growth and/or colony formation in soft agar ortubular network formation in three-dimensional basement membrane orextracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials. Further, any assays known tothose skilled in the art can be used to evaluate the prophylactic and/ortherapeutic utility of the combinatorial therapies disclosed herein fortreatment or prevention of cancer, inflammatory disorder, or autoimmunedisease.

D. HIV Infection

The Human Immunodeficiency Virus (HIV) infects millions of peopleglobally. Cases are reported from nearly every country amounting to 40million adults and children living with HIV/AIDS worldwide. In 2001, 5million people were newly infected with HIV, and there were 3 millionadult and child deaths due to HIV/AIDS. A full third of those peopleliving with AIDS are aged 15-24 (World Health Organization, 2001).

The typical human immune system response, killing the invading virion,is taxed because the virus infects and kills the immune system's Tcells. In addition, viral reverse transcriptase, the enzyme used inmaking a new virion particle, is not very specific, and causestranscription mistakes that result in continually changed glycoproteinson the surface of the viral protective coat. This lack of specificitydecreases the immune system's effectiveness because antibodiesspecifically produced against one glycoprotein may be useless againstanother, hence reducing the number of antibodies available to fight thevirus. The virus continues to reproduce while the immune response systemcontinues to weaken. Eventually, the HIV largely holds free reign overthe body's immune system, allowing opportunistic infections to set inand, without the administration of antiviral agents, immunomodulators,or both, death may result.

While treatments for HIV/AIDS exist, the drugs currently used intreatment modalities exhibit numerous side effects, require prolongedtreatment that often induces drug resistance, and do not result incomplete eradication of the virus from the body. For example, nucleosideanalogs, such as 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxycytidine(ddC), 2′,3′-dideoxythymidinene (d4T), 2′,3′-dideoxyinosine (ddI), and2′,3′-dideoxy-3′-thia-cytidine (3TC) have been shown to be relativelyeffective in halting HIV replication at the reverse transcriptase (RT)stage. Even with the current success of reverse transcriptaseinhibitors, it has been found that HIV patients can become resistant toa single inhibitor. Thus, it is desirable to develop compounds for usein combination with other known HIV treatments to further combat HIVinfection and inhibit the replication of drug resistant strains of HIV.

In use, the locked dimer of the present invention may be administered incombination with one or more other compound having activity against HIVdisease or HIV-related disease. By “HIV disease or HIV-related disease”we mean a disease state which is marked by HIV infection. Such disordersassociated with HIV infection include, but are not limited to, AIDS,Kaposi's sarcoma, opportunistic infections such as those caused byPneumocystis carinii and Mycobacterium tuberculosis; oral lesions,including thrush, hairy leukoplakia, and aphthous ulcers; generalizedlymphadenopathy, shingles, thrombocytopenia, aseptic meningitis, andneurologic disease such as toxoplasmosis, cryptococcosis, CMV infection,primary CNS lymphoma, and HIV-associated dementia, peripheralneuropathies, seizures and myopathy.

Standard tissue culture models of HIV infection can be used to determinethe efficacy of CXCL12α₂ locked dimer as an HIV entry inhibitor incombination with current HIV entry inhibitors. Suitable therapeuticagents for use in combination with the compounds of the presentinvention include, but are not limited to, protease inhibitors,non-nucleoside reverse transcriptase inhibitors, nucleoside reversetranscriptase inhibitors, antiretroviral nucleosides, entry inhibitorsas well as other anti-viral agents effective to inhibit or treat HIVinfection. Further examples of suitable therapeutic agents include, butare not limited to, zidovudine, didanosine, stavudine, interferon,lamivudine, adefovir, nevirapine, delaviridine, loviride, saquinavir,indinavir and AZT. Other suitable therapeutic agents include, but arenot limited to, antibiotics or other anti-viral agents, e.g., acyclovir.Other combination therapies known to those of skill in the art can beused in conjunction with the compositions and methods of the presentinvention.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference for all purposes.

It is understood that the invention is not confined to the specificreagents, formulations, reaction conditions, etc., herein illustratedand described, but embraces such modified forms thereof as come withinthe scope of the following claims.

REFERENCES

-   1. Allen et al. (2007). Annu Rev Immunol 25, 787-820.-   2. Shirozu et al. (1995). Genomics 28, 495-500.-   3. Doranz et al. (1999). J Virol 73, 2752-2761.-   4. Nagasawa et al. (1994). Proc Natl Acad Sci USA 91, 2305-2309.-   5. Nagasawa et al. (1996). Nature 382, 635-638.-   6. Tachibana et al. (1998). Nature 393, 591-594.-   7. Zou et al. (1998). Nature 393, 595-599.-   8. Heidemann et al. (2004). Am J Physiol Gastrointest Liver Physiol    286, G1059-1068.-   9. Bleul et al. (1996). Nature 382, 829-833.-   10. Feng et al. (1996). Science 272, 872-877.-   11. D'Souza et al. (1996). et al. Nat Med 2, 1293-1300.-   12. D'Souzav et al. (2000). JAMA 284, 215-222.-   13. Miedema et al. (1994). Immunol Rev 140, 35-72.-   14. Mulle et al. (2001). Nature 410, 50-56.-   15. Zlotnik, A. (2006). Int J Cancer 119, 2026-2029.-   16. Veldkamp et al. (2006). J Mol Biol 359, 1400-1409.-   17. Farzan et al. (2002). J Biol Chem 277, 29484-29489.-   18. Farzan et al. (1999). Cell 96, 667-676.-   19. Farzan et al. (2002). J Biol Chem 277, 40397-40402.-   20. Seibert et al. (2002). Proc Natl Acad Sci USA 99, 11031-11036.-   21. Fong et al. (2002). J Biol Chem 277, 19418-19423.-   22. Babcock et al. (2003). J Biol Chem 278, 3378-3385.-   23. Veldkamp et al. (2005). Protein Sci 14, 1071-1081.-   24. Segers et al. (2007). Circulation 116, 1683-1692.-   25. O'Boyle et al. (2008). Examination of the chemotaxis blockade by    non-GAG-binding chemokine receptor agonists. In Keystone Conference    on Chemokines and Chemokine Receptors. Keystone Symposia, Keystone    Resort, Keystone, Co.-   26. Vijayalakshmi et al. (1994). Protein Sci 3, 2254-2271.-   27. Somers et al. (2000). Cell 103, 467-479.-   28. Veldkamp et al. (2007). Protein Expr Purif 52, 202-209.-   29. Fricker et al. (2006). Biochem Pharmacol 72, 588-596.-   30. Milligan et al. (2007). Trends Pharmacol Sci 28, 615-620.-   31. Chabre et al. (2005). Biochemistry 44, 9395-9403.-   32. James et al. (2006). Nat Methods 3, 1001-1006.-   33. El-Asmar et al. (2005). Mol Pharmacol 67, 460-469.-   34. Springael et al. (2005). Cytokine Growth Factor Rev 16, 611-623.-   35. Bouvier et al. (2007). Nat Methods 4, 3-4; author reply 4.-   36. Rosenbaum et al. (2007). Science 318, 1266-1273.-   37. Cherezov et al. (2007). Science 318, 1258-1265.-   38. Crump et al. (1997). Embo J 16, 6996-7007.-   39. Loetscher et al. (1998). J Biol Chem 273, 22279-22283.-   40. Jin et al. (2007). J Biol Chem. September 21; 282(38):27976-83.-   41. Markley et al. (2003). Methods Biochem Anal 44, 89-113.-   42. Peterson et al. (2006). J Mol Biol 363, 137-147.-   43. Cornilescu et al. (1999). J Biomol NMR 13, 289-302.-   44. Herrmann et al. (2002). J Mol Biol 319, 209-227.-   45. Linge et al. (2003). Proteins 50, 496-506.-   46. Ohnishi et al. (2000). J Interferon Cytokine Res 20, 691-700.-   47. Princen et al. (2003). Cytometry 51A, 35-45.-   48. Heveker et al. (1998). Curr Biol 8, 369-376.-   49. Peterson et al. (2006). J Mol Biol 363, 813-822.-   50. Williams et al. (1996). J Biol Chem 271, 9579-9586.-   51. Leong et al. (1997). Protein Sci 6, 609-617.-   52. Fernando et al. (2004). J Biol Chem 279, 36175-36178.-   53. Rajarathnam et al. (2006). Biochemistry 45, 7882-7888.-   54. Schnitzel et al. (1994). J Leukoc Biol 55, 763-770.-   55. Moore, M. A. (2001). Bioessays 23, 674-676.-   56. Murdoch, C. (2000). Immunol Rev 177, 175-184.-   57. Liotta, L. A. (2001). Nature 410, 24-25.-   58. Helbig et al. (2003). J Biol Chem 278, 21631-21638.-   59. Cooper et al. (2003). Cancer 97, 739-747.-   60. Taichman et al. (2002). Cancer Res 62, 1832-1837.-   61. Zeelenberg, et al. (2003). Cancer Res 63, 3833-3839.-   62. Moller et al. (2003). Leukemia 17, 203-210.-   63. Murakami et al. (2002). Cancer Res 62, 7328-7334.-   64. Payne et al. (2002). J Invest Dermatol 118, 915-922.-   65. Delilbasi et al. (2004). Oral Oncol 40, 154-157.-   66. Scotton et al. (2002). Cancer Res 62, 5930-5938.-   67. Hall et al. (2003). Mol Endocrinol 17, 792-803.-   68. Milliken et al. (2002). Clin Cancer Res 8, 1108-1114.-   69. Burger et al. (2003). Oncogene 22, 8093-8101.-   70. Phillips et al. (2003). Am J Respir Crit Care Med 167,    1676-1686.-   71. Mori et al. (2004). Mol Cancer Ther 3, 29-37.-   72. Gockel et al. (2007). Future Oncol 3, 119-122.-   73. Zlotnik, A. (2006). Contrib Microbiol 13, 191-199.-   74. Retz et al. (2005). Int J Cancer 114, 182-189.-   75. Eisenhardt et al. (2005). Eur Urol 47, 111-117.-   76. Wang et al. (2006). Cancer Metastasis Rev 25, 573-587-   77. Burger, J. A. (2008) Leuk Res.-   78. Burger et al. (2008) Leukemia.-   79. Nervi et al. (2008) Blood.-   80. Tavor et al. (2008) Leukemia 22, 2151-5158.-   81. Azab et al. (2009) Blood.-   82. Burger et al. (2006). Blood 107, 1761-1767.

We claim:
 1. A CXCL12-α₂ locked dimer polypeptide, wherein the dimercomprises two monomers locked together.
 2. The dimer of claim 1 whereinthe monomers are not identical.
 3. The dimer of claim 1 wherein themonomers are identical.
 4. The dimer of claim 1 wherein at least one ofthe monomers has the amino acid sequence as shown in SEQ ID NO:1.
 5. Thedimer of claim 1 wherein the monomers are locked together at residuesL36 and A65 of SEQ ID NO:
 1. 6. A composition comprising the dimer ofclaim 1, and a pharmaceutically acceptable carrier or diluent.
 7. Anisolated CXCL12-α₂ locked dimer polypeptide, wherein the dimer consistsof two monomers locked together.
 8. The dimer of claim 7 wherein atleast one of the monomers has the amino acid sequence as shown in SEQ IDNO:1.
 9. A kit comprising a CXCL12-α₂ locked dimer polypeptide whereinthe dimer comprises monomers having the amino acid sequence as shown inSEQ ID NO:1, a pharmaceutically acceptable carrier or diluent, andinstructional material.